1. Field of the Invention
The present invention relates to a reagent for determining the quantity of creatine kinase (hereafter abbreviated as CK) in living body fluids.
2. Related Art Statement
CK occurs in the systemic muscular tissues and brain of a human body and, in the field of clinical examinations, is one of the important items needed for usual diagnosis of muscular diseases, nervous disorders, central nervous system disorders, mental troubles, heart troubles and the like.
CK is an enzyme that catalyzes reversibly the reaction expression in terms of the following formula 1: ##STR1## wherein CP represents creatine phosphate, C represents creatine, ADP represents adenosine diphosphate, and ATP represents adenosine triphosphate.
Heretofore, various methods for the measurement of CK has been proposed. One type of CK measurement is described as the measurement of activity in the lefthand direction in Formula 1, including (i) the measurement of inorganic phosphoric acid formed by hydrolysis of CP, (ii) the derivation of ADP to reduction type beta-nicotinamide adenine dinucleotide (hereinafter abbreviated as NADH) under the action of pyruvate kinase (hereinafter abbreviated as PK) and lactose dehydrogenase (hereinafer abbreciated as LDH) for the determination of absorption losses, and (iii) the derivation of ADP to pyruvic acid with PK, which is in turn allowed to react with 2,4-dinitrophenylhidrazine to measure the resulting hidrazone. The other type is described as the measurement of activity in the right hand direction in Formula 1, including (i) the reaction of the resulting C with a pigment of colorimetry or fluorometry, (ii) the use of luciferase (see Japanese Patent Laid-Open Specification Nos. 51-41597, 55-120796, 56-26200 and 57-105199), (iii) the use of phosphoglyceric acid kinase (hereinafter abbreviated as PGK) and glyceraldehyde-3-phosphate dehydrogenase (hereinafter abbreviated as GAPDH) (see Japanese Patent Publication No. 59-34119 and Japanese Patent Laid-Open Specification No. 56-155000) and (iv) the use of hexokinase (hereinafter abbreviated as HK) and glucose-6-phosphate dehydrogenase (hereinafter abbreviated as G6PDH). The combined HK/G6PDH method is most frequently used, since it is the most excellent in principle, shows improved sensitivity and reproducibility, and can treat a multiplicity of samples.
Incidentally, since CK is an unstable enzyme, the so-called SH group-containing compound (hereinafter called simply the SH reagent) such as N-acetylcysteine (hereinafter abbreviated as NAC), dithiothreitol, glutathione, mercaptoethanol and the like is ordinarily used as the activator for the purpose of enhancing the activation effect of the activity of CK, when its activity value is measured. Reportedly, particular preference is given to NAC (see J. Clin. Chem. Clin. Biochem Vol. 15, pp. 249.about.254, 1977). The divalentions most preferably used in the reactions on which CK acts are magnesium ions. It is noted that in this connection that some literature reports that, in the reactions of Formula 1 on which CK acts, the km value of magnesium ions is Mg.sup.++ 6.times.10.sup.-4 M for the left hand direction and Mg.sup.++ 6.times.10.sup.-3 M for the right hand direction (see "Clinical Examinations", Vol. 15, No. 12, pp. 1257, 1971). Thus, in addition to the primary components, NAC and magnesium ions are considered to be the ingredients which are essentially and simultaneously required to preferably effect the reactions on which CK acts.
Heretofore, the reagents for the measurement of CK have had the disadvantages that they are poor in storage stability at room temperature in the state of a solution and, in particular, their service life is very short at room temperature (18.degree..about.37.degree. C.). Consequently, it has been found that, since such reagents should be used within a short period of time just after preparation, their use is inefficient. For example, in view of the fact that they should individually be prepared whenever a number of samples are treated over a prolonged period. Alternatively, they are unsuitable for use with an emergency automatic analyzer designed to examine a limited number of unexpected but extremely urgent samples. The incorporation of NAC in particular serves to enhance the activation effect on the activity of CK. Since NAC is a relatively unstable reagent, however, the SH group contained therein is gradually oxidized in the state of solution to such an extent that the sufficient activation effect on the activity of CK is no longer sustained. It is further reported that the decomposed product of NAC tends to inhibit the activity of CK (see CLIN. CHEM. Vol. 22, No. 5, pp. 650.about.656, 1976). Combined with a reduction in the activation effect, this is greatly responsible for the unstableness of the reagent for CK measurement in the state of a solution.
A concrete phenomenon resulting from the unstableness of NAC occurs noticeably when, for instance, serum dilution testing is carried out. To put it more concretely, there is a gradual decrease in the gradient of the calibration curve obtained from the serum dilution testng with the lapse of days after the preparation of reagents, say, a lowering of sensitivity. Clearly, such a phenomenon poses a grave problem in view of accuracy control in clinical examinations.
It is reported, on the other hand, that NAC is stabilized by a chelating agent such as ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA), and the resulting NAC is kept stable for about 7 days under storage testing at 25.degree. C. However, even such a treatment results in unavoidable occurrence of a factor inhibiting the activity of CK originating from NAC. In practice, the CK activity value achieved with the use of such a reagent for CK measurement remains constant for about two days, when the prepared reagent is stored at 25.degree. C., but later decreases sharply. That is to say, the conclusion that the effect of EDTA upon the stabilization of NAC does not yet reach any practical level can hardly be avoided.